The nervous system uses neural circuits to generate cognition and behavior. These circuits are often disrupted in spinal cord injury or stroke, and are aberrant in mental disorders. One way to treat these conditions is to re- grow or modify neural circuits. Thus, understanding how neural circuits are assembled may aid in the design of regenerative therapies, and brain stimulation therapies to treat mental disorders. During circuit assembly, nervous systems exhibit patterned spontaneous network activity (PaSNA). PaSNA is necessary to build a functional circuit in vertebrates and invertebrates, yet we know surprisingly little about the mechanisms involved. My strategy is to use Drosophila embryonic and larval locomotion as a system to uncover the underlying molecular and cellular mechanisms by which PaSNA promotes neural circuit assembly. The advent of new tools in Drosophila, such as optogenetic reagents, Gal4 lines to manipulate single neurons in the central nervous system (CNS), and a transmission electron microscopy connectome of the entire CNS make this a powerful system for studying neural circuits. Similar to vertebrates, premotor neurons are required for PaSNA. This raises the questions that this proposal addresses: Which specific interneuron populations generate PaSNA, do different interneurons have different patterns of activity, and how does interneuron PaSNA drive motor circuit development? With the use of Gal4 lines unique to our system and genetically encoded calcium indicators, I will identify the interneurons participating in PaSNA. I will use a characterized circuit required for larval locomotion together with optogenetic tools to identify the effect of PaSNA in establishing circuit connectivity and function. This proposal will reveal (1) the neuronal components that generate PaSNA during locomotor circuit assembly, (2) whether PaSNA is stochastic or stereotypic with the population of active neurons, (3) whether there is a correlation between neuronal identity and participation in PaSNA; (4) whether PaSNA is required for locomotor circuit establishment, and (5) whether PaSNA is required for locomotor circuit function. These represent critical advances that will provide insight into the formation and function of neural circuits in both Drosophila and mammals, where PaSNA is a conserved element of neural circuit formation.